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Biomedical subjects

P Wilhelm

Publications and source records attributed to P Wilhelm.

At least 19 recordsLinked to original sources
PubMed2006

Microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study.

Over recent years, the use of individually ventilated cage (IVC) rack systems in laboratory rodent facilities has increased. Since every cage in an IVC rack may be assumed to be a separate microbiological unit, comprehensive microbiological monitoring of animals kept in IVCs has become a challenging task, which may be addressed by the appropriate use of sentinel mice. Traditionally, these sentinels have been exposed to soiled bedding but more recently, the concept of exposure to exhaust air has been considered. The work reported here was aimed firstly at testing the efficiency of a sentinel-based microbiological monitoring programme under field conditions in a quarantine unit and in a multi-user unit with frequent imports of mouse colonies from various sources. Secondly, it was aimed at determining biocontainment of naturally infected mice kept in an IVC rack, which included breeding of the mice. Sentinels were exposed both to soiled bedding and to exhaust air. The mice which were used in the study carried prevalent infectious agents encountered in research animal facilities including mouse hepatitis virus (MHV), mouse parvovirus (MPV), intestinal flagellates and pinworms. Our data indicate that the sentinel-based health monitoring programme allowed rapid detection of MHV, intestinal flagellates and pinworms investigated by a combination of soiled bedding and exhaust air exposure. MHV was also detected by exposure to exhaust air only. The IVC rack used in this study provided biocontainment when infected mice were kept together with non-infected mice in separate cages in the same IVC rack.

Animal Husbandry↗
PubMed2005

On-line tracking of the coating of nanoscaled silica with titania nanoparticles via zeta-potential measurements.

Nanoscaled spherical silica particles were directly coated with the titania nanoparticles by means of a heterogenic coagulation. Silica was prepared by the Stöber method, titania by a hydrolysis-condensation reaction of tetrapropylorthotitanate under acidic conditions. The on-line tracking of the coating process was performed by measuring the change in zeta potential during the gradual addition of a titania sol to the spherical silica particles. Silica particles of various sizes were used to determine the consumption of the titania sol in the dependence upon the particle size. The coated and uncoated particles were characterized by zeta-potential measurements, acoustic attenuation spectroscopy, dynamic light scattering, and scanning electron microscopy.

Journal Article↗
PubMed2005

Performance evaluation of IVC systems.

An expert Working Group was set up in December 2000 to develop recommendations for users and industry on the evaluation of proper function and operation of individually ventilated cage (IVC) systems. The full report of their recommendations is in two parts--'Part 1: Test Instructions' and 'Part 2: Evaluation Criteria'--both of which have been published in full on the Laboratory Animals Ltd website. They can be found at http://www.lal.org.uk/IVC/index.html. Evaluation of and feedback on the recommendations to further refine their use and scientific basis is encouraged. This Summary Report provides a brief overview of the background to the development of the full report and the issues it addresses.

Animal Welfare↗
PubMed2005

Monitoring a mouse colony for Helicobacter bilis using a Helicobacter-genus-specific nested PCR.

Although Helicobacter infections of laboratory mice are usually subclinical, they may interfere with in vivo experiments and thus may lead to misinterpretation of data. As such, it is important to provide a means to unequivocally identify infections with murine Helicobacter spp. In the present study, a nested polymerase chain reaction (PCR) was established and shown to be 10 to 100 times more sensitive than the single-step PCR commonly used for routine diagnosis of Helicobacter spp. Experimental infection of Helicobacter-free mice demonstrated that faeces, caecum, colon and rectum but not liver are equally suitable for the detection of H. bilis. However, use of faecal pellets is advantageous since detection of H. bilis is possible one week after infection and analysis of faeces instead of tissues avoids euthanasia of animals. Furthermore, it generates representative data for all animals housed in the same cage and analysis can be repeatedly performed. Use of samples from breeding pairs but not offspring provides representative information about the Helicobacter status of a mouse colony. Both C3H/HeJ and C57BL/6 mice appear to be susceptible to H. bilis and persistent infection was observed during the 20-week experimental period. Analysis of pooled faecal pellets by nested PCR seems to be the most sensitive approach for H. bilis monitoring of the given breeding colony.

Animals↗
PubMed2001

Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF.

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-gamma, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF(-/-) (B6.TNF(-/-)) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF(-/-) mice mounted an L. major-specific IFN-gamma response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF(-/-) inflammatory macrophages released 10-fold less NO in response to IFN-gamma than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF(-/-) macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF(-/-) mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.

Animals↗
PubMed2000

Tumor necrosis factor sustains the generalized lymphoproliferative disorder (gld) phenotype.

Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF(-/-) mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.

Animals↗
PubMed1993

Grading cervical dysplasia with AgNORs using a semiautomated image analysis system.

Colposcopic biopsies were classified according to previously established criteria by a group of three pathologists interested in cervical pathology. Ten cases were identified in each of the following five groups: normal, koilocytosis, low grade squamous intraepithelial lesions (CIN 1), high grade squamous intraepithelial lesions (CIN 2) and high grade squamous intraepithelial lesions (CIN 3). The Crocker technique was used to stain the sections cut 3 microns thick. With ths silver stain the nucleolar organizer regions (NORs) are stained black and referred to as AgNORs. It has been shown that malignant and premalignant changes in cells produce an increase in AgNORs. In each case eight images were captured using a 100x oil-immersion objective and stored in a Datacube Maxvideo system as 512 x 480 pixels in an 8-bit grayscale per image. The images were processed using the NeoPath field-of-view computer to detect the AgNORs and nuclei by using grayscale mathematical morphology algorithms. Color overlays of the AgNORs and nuclei were created using segmentation algorithms. The results show that it is possible to differentiate between low grade squamous intraepithelial lesions (CIN 1) and high grade squamous intraepithelial lesions (CIN 2 and CIN 3) taken together; however, there is no difference between low grade squamous intraepithelial lesions (CIN 1) and koilocytosis. The results support the concept that dysplasia cannot be classified effectively into three grades and that low grade squamous intraepithelial lesions (mild dysplasia [CIN 1]) is indistinguishable from koilocytosis.

Analysis of Variance↗
PubMed1987

Epitope localization in antigen-monoclonal-antibody complexes by small-angle X-ray scattering. An approach to domain organization in the beta 2 subunit of Escherichia coli tryptophan synthase.

Each polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) is made of two domains, F1 (N-terminal) and F2 (C-terminal). To determine the relative position of these domains in the native protein, complexes between beta 2 and Fab fragments from two monoclonal antibodies, one specific for F1 (68-1) and the other for F2 (93-6), have been prepared and purified. Small-angle X-ray scattering measurements have been made on solutions of each complex. From the experimental scattering curves obtained, computer modeling leads to structural models of the two beta 2-Fab complexes. Though relatively low, the resolution of these models allows the localization on beta 2 of the antigenic sites recognized by the two antibodies, to show that the C-terminal F2 domains lie at the distal ends of the elongated beta 2 protein, and to show how steric hindrance prevents beta 2, though structurally and functionally dimeric, from binding more than one Fab 93-6 fragment per dimer.

Antigen-Antibody Complex↗
PubMed1982

Small-angle X-ray scattering studies of tryptophan synthase from Escherichia coli and its alpha and beta 2 subunits.

The alpha and beta 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering. The molecular parameters are: radius of gyration, alpha: 1.95 nm, beta 2: 3.01 nm; maximum particle diameter, alpha: 5.8 nm, beta 2: 10.5 nm; and hydrated volume, alpha: 60 nm3, beta 2 160 nm3. The shape of the alpha subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the beta 2 subunits. The alpha 2 beta 2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the alpha and beta 2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the alpha 2 beta 2 complex.

Bacterial Proteins↗
PubMed1978

Small-angle X-ray studies of a human immunoglobulin M.

The conformation of a Waldenström immunoglobulin M (IgM) with antibody-like activity for X-ray contrast media, based on 3-amino-2,4,6-triiodobenzoic acid, was studied by small-angle X-ray scattering. The radius of gyration was determined as 12.1 nm, the maximum distance 35 nm, the volume 1900 nm3. A flat star-shaped model was found to be equivalent in scattering. Aggregation of IgM molecules seems to take place as side-by-side combinations of single molecules, manifesting itself as a relatively large increase of the radius of gyration and unchanged thickness of the flat aggregates.

Humans↗
PubMed1978

Effect of prostaglandins on the blood-aqueous barrier of the perfused cat eye.

With a technique of direct visualization of the arterially perfused cat eye, the pressure head may be chosen so that fluorescein added to the perfusate just barely stains the ciliary processes. After addition of PGE1, PGE2, PGF2alpha arachidonic acid, or indomethacin, with or without PG's, no more dye emerged from the processes. The addition of acetylcholine in the eserinized eye floods the processes with dye, apparently affecting the pore size of the blood-aqueous barrier; PG's may slightly inhibit, rather than facilitate, the emergence of fluorescein from the processes. It is speculated that in the cat PG's elevate pressure and protein content by backflow from the circle of Hovius, which is the equivalent of Schlemm's canal.

Acetylcholine↗